Respiratory syncytial virus (RSV) is an important human respiratory pathogen, particularly for infants and older adults (Falsey and Walsh, 2000; Heilman, 1990). It is a single strand, non-segmented, negative sense RNA virus in the Mononegavirales superfamily, the Paramyxoviridae family, and the Pneumovirinae subfamily. All of the paramyxoviruses enter target cells by fusion at neutral pH, mediated by a similar fusion (F) protein and replicate entirely in the cytoplasm. The F protein expression in infected cells is also responsible for the typical syncytial pathology caused by RSV, the generation of multinucleated giant cells. RSV differs from viruses in the other Paramyxoviridae subfamily, the Paramyxovirinae, in several ways: its genome contains 10 genes, instead of 6 or 7; it has two nonstructural protein genes (NS1 and NS2) that precede the nucleocapsid (N) gene; and its attachment-enhancing protein, G (glycoprotein), is highly decorated with O-linked carbohydrate chains (Collins, Mcintosh, and Chanock, 2001).
The 15.2 kb RSV genome replicates and expresses its genes in the cytoplasm of host cells. It is encased in a helical nucleocapsid structure provided by the N protein. This nucleocapsid complex is used by the viral polymerase as the template for transcription of mRNA and for production of full-length antigenomic copies. These antigenomes are also encased in helical nucleocapsid structures by the N protein and are copied by the polymerase to produce genomes.
The major polymerase subunit is the L (large) protein, but the P (phosphoprotein) is also required for polymerase activity. While the L and P proteins are adequate for genome and antigenome synthesis, a third component, the M2-1 protein, is required for transcription of mRNA. It prevents premature termination during transcription of long mRNAs (Collins et al., 1996). Transcription of each gene initiates at a conserved 10 nt gene start (GS) sequence and terminates at a relatively conserved gene end (GE) sequence (Collins, McIntosh, and Chanock, 2001). M2-2, a second protein encoded by the M2 mRNA controls the balance between transcription and replication (Bermingham and Collins, 1999).
The viral F, G and SH (small hydrophobic) glycoproteins are synthesized in the endoplasmic reticulum and transit through the Golgi where the F protein is cleaved in two places by a furin-like enzyme (Zimmer, Budz, and Herrier, 2001) to reach the cell surface. Once at the cell surface, the F protein can cause the membranes of infected immortalized cells in culture to fuse with those of neighboring cells, producing multinucleated giant cells called syncytia. Syncytia formation does not require the G protein, but is enhanced by its presence (Karron et al., 1997; Techaarpornkul, Barretto, and Peeples, 2001). Syncytia formation is also thought to be the main cytopathic effect caused by RSV, and the main reason that RSV-infected cells die. RSV infection of cultured immortal cells does not lead to an early shutdown of protein synthesis (Levine, Peeples, and Hamilton, 1977) as do other, highly cytotoxic negative strand viruses.
Interestingly, RSV inoculation of cultured well-differentiated human airway epithelial (WD-HAE) cells results in infection of only the ciliated cells at the superficial surface but infection does not result in syncytia or in rapid cell death (Zhang et al., 2002). More recent experiments have demonstrated that these infected cells are killed by RSV infection, but that this process takes 5 to 7 days (Zhang, L, Peeples, M. E., Collins, P. L., and Pickles, R., unpublished data), indicating that RSV infection is slowly toxic for these cells even in the absence of syncytia formation.
Virion assembly takes place at the plasma membrane, orchestrated by the matrix (M) protein and culminating in the budding and release of progeny virions. In addition to their roles in virion infectivity, the G protein has been shown to enhance release of virions from infected cells (Techaarpornkul, Barretto, and Peeples, 2001). In other paramyxoviruses, and the F protein has also been shown to be important for budding (Cathomen, Naim, and Cattaneo, 1998; Russell, Jardetzky, and Lamb, 2001; Schmitt et al., 2002; Waning et al., 2002). However, RSV, like other negative strand RNA viruses, should not require its glycoproteins for genome replication and gene expression.
Non-cytotoxic replicons have previously been generated for positive strand RNA viruses, such as Sindbis and hepatitis C virus, by removing the viral glycoprotein and capsid genes (Blight, Kolykhalov, and Rice, 2000; Frolov et al., 1999; Lohmann, 1999). Replication proteins did not need to be supplied separately for these positive strand viruses because the positive strand genome is also an mRNA and is translated to produce these proteins.
The replicon produces all of the RSV proteins except for its glycoproteins. To mobilize a replicon from a cell line in which it is replicating, a viral glycoprotein gene, the vesicular stomatitis virus G gene, was transfected into the replicon-containing cell line. The G glycoprotein complements the RSV replicon, allowing the formation and secretion of replicon/virions. The medium can be used to inoculate fresh cells, which in the presence of blasticidin, form colonies of replicon-containing cells.